This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background

The pathogenic mold Aspergillus fumigatus is the most frequent infectious cause of death in severely immunocompromised individuals
such as leukemia and bone marrow transplant patients. Germination of inhaled conidia
(asexual spores) in the host is critical for the initiation of infection, but little
is known about the underlying mechanisms of this process.

Results

To gain insights into early germination events and facilitate the identification of
potential stage-specific biomarkers and vaccine candidates, we have used quantitative
shotgun proteomics to elucidate patterns of protein abundance changes during early
fungal development. Four different stages were examined: dormant conidia, isotropically
expanding conidia, hyphae in which germ tube emergence has just begun, and pre-septation
hyphae. To enrich for glycan-linked cell wall proteins we used an alkaline cell extraction
method. Shotgun proteomic resulted in the identification of 375 unique gene products
with high confidence, with no evidence for enrichment of cell wall-immobilized and
secreted proteins. The most interesting discovery was the identification of 52 proteins
enriched in dormant conidia including 28 proteins that have never been detected in
the A. fumigatus conidial proteome such as signaling protein Pil1, chaperones BipA and calnexin, and
transcription factor HapB. Additionally we found many small, Aspergillus specific
proteins of unknown function including 17 hypothetical proteins. Thus, the most abundant
protein, Grg1 (AFUA_5G14210), was also one of the smallest proteins detected in this
study (M.W. 7,367). Among previously characterized proteins were melanin pigment and
pseurotin A biosynthesis enzymes, histones H3 and H4.1, and other proteins involved
in conidiation and response to oxidative or hypoxic stress. In contrast, expanding
conidia, hyphae with early germ tubes, and pre-septation hyphae samples were enriched
for proteins responsible for housekeeping functions, particularly translation, respiratory
metabolism, amino acid and carbohydrate biosynthesis, and the tricarboxylic acid cycle.

Conclusions

The observed temporal expression patterns suggest that the A. fumigatus conidia are dominated by small, lineage-specific proteins. Some of them may play
key roles in host-pathogen interactions, signal transduction during conidial germination,
or survival in hostile environments.

Keywords:

Background

Aspergillus fumigatus is the most common airborne fungal pathogen, which can infect ever increasing numbers
of patients with lung disease, immune system disorders or undergoing immunosuppression
therapy [1]. In patients with asthma and cystic fibrosis, it can cause allergic diseases like
allergic bronchopulmonary aspergillosis. In immunosuppressed individuals such as leukemia
and bone marrow transplant patients, inhalation of A. fumigatus conidia (asexual spores) can cause invasive aspergillosis (IA), a life-threatening
disease, which is difficult to diagnose and treat. If successful in reaching the innate
immune defense in the lungs, conidia germinate into hyphae, long finger-like projections
that invade host tissues and blood vessels within days or even hours after colonization.
Despite the importance of the early morphogenetic transition for initiation of infection,
its specific mechanisms are not all well-understood, which hinders the development
of better diagnostic and therapeutic approaches to combat IA.

The availability of two sequenced A. fumigatus genomes, AF293 and A1163 [2,3], have enabled high-throughput transcriptomic and proteomic approaches and, thus,
greatly facilitated the pace of discovery of new biomarkers and therapeutic targets
for IA. Previous proteomic studies have identified a number of proteins involved in
early stages of A. fumigatus development and early interactions with the human host [4]. Traditionally proteomic studies rely on gel-based separations such as 2-dimensional
polyacrylamide gel electrophoresis (2-D PAGE) followed by mass spectrometry (MS).
The methods helped to identify reactive oxygen detoxification enzymes, pigment biosynthesis
enzymes and other highly abundant proteins in the A. fumigatus conidial proteome [5-8].

While 2D gel approaches can identify proteins in their intact forms, they lack sufficient
sensitivity and dynamic range for protein quantification. Furthermore, 2D gels regularly
fail to resolve proteins with physicochemical characteristics such as high hydrophobicity,
extreme pI and Mr values and covalent attachment to membranes or cell walls [9]. As a result, little is known about expression status and functional roles of such
proteins. Quantitative shotgun proteomics based on liquid chromatography tandem MS (LC-MS/MS)
holds promise to more comprehensive proteome surveys, including comparative analyses
of proteins from different developmental stages [10]. Recently, we (SC and DP) profiled A. fumigatus early development proteome states using shotgun proteomics based on isobaric tagging
of peptides (iTRAQ), accompanied by a simultaneous transcriptome analysis [11]. This approach resulted in identification of 231 proteins with high confidence. The
current study also aims to survey the A. fumigatus early developmental proteome, although it is focused on earlier time points and involves
different growth conditions. Although the hydrophobin RodA was among the most abundant
proteins, the attempt to enrich for cell wall-immobilized proteins was apparently
not successful. Using a shotgun proteomics approach, 375 proteins were identified
including 207 proteins that have not been detected using iTRAQ shotgun proteomics
[11]. Additionally we found 28 dormant conidia-enriched proteins that have not been previously
detected in the A. fumigatus proteomes of conidia or pre-septation hyphae.

Results and discussion

Selection of time points that represent distinct stages of early fungal development
for proteomics analysis

In most fungi, conidial dormancy is controlled by exogenous factors such as the availability
of moisture, oxygen and nutrients [12]. It has been established that, when inoculated to culture medium containing a carbon
source, A. fumigatus conidia synchronously break dormancy and begin nuclear division and morphological
development. Nuclear division and morphological development remain roughly synchronous
for at least 12 h, a time that encompasses the first several rounds of mitosis and
early developmental landmarks [13]. In this study, we exploited this inherent synchrony to characterize the A. fumigatus proteome during the early stages of fungal development.

To select specific stages for proteomics analysis, A. fumigatus conidia were inoculated in glucose complete medium and sequential samples were examined
microscopically for developmental landmarks every 30 min. As summarized in Figure
1, the conidium expands isotropically for 4 h at 37°C in complete medium. Most cells
polarize and send out the first germ tube between 5 and 6 h and continue to elongate
becoming hyphae. The first septum forms near the base of the hypha between 9 and 10 h,
asymmetrically dividing the hypha into two compartments. At about the same time, the
first branch forms on the apical side of the septum. Hyphae continue to elongate and
branch and eventually form a mycelial mat. Based on the microscopy data, four time
points were selected for proteomics analysis: dormant conidia (0 h), isotropically
expanding conidia (4 h), hyphae with early germ tubes (6 h), and pre-septation hyphae
(8 h).

Figure 1.A. fumigatusearly development stages selected for proteomic analysis. For the 0 h time point dormant conidia were stained with Calcofluor and Hoechsts.
For the 4, 6 and 9 h time points, 3 x 106 spores were inoculated into 10 mL of GMM and incubated at 37°C. The samples were
fixed and stained with Calcofluor and Hoechsts. Upper panel shows DIC image lower
panel shows stained florescent image. All pictures were taken at 100X magnification.
Scale bar = 3 μm.

Although A. fumigatus conidia were grown in vitro, we believe that these four time points represent critical developmentally-matched
stages of fungal growth in vivo. Previous work has shown that the cell wall of A. fumigatus is organized in domains that change during early growth [14]. It is likely that some of this change is associated with new proteins being added
to the wall at different stages of development as well as by reorganization and modification
of proteins within the wall. Though the timeline of A. fumigatus development within human hosts is not known, we can extrapolate from the in vitro data cited above and in mouse model systems [15].

A. Fumigatus proteins expressed during early fungal development

A. fumigatus proteins were extracted using a mild alkali method. They were analyzed using LC-MS/MS
followed by a modified spectral counting technique, called APEX [16]. Using this approach, we detected 570 unique proteins which represented 5.6% of the
predicted A. fumigatus proteome. The estimated sequence coverage for proteins ranged from 4% to 100%, and
Mascot scores ranged from 40 to 4,978. Theoretical isoelectric points (pIs) varied between 3.8 and 11.8, and molecular masses (Mr) between 6,287 and 234,143 Da. Experimentally observed proteins were mapped against
the theoretical proteome in relation to Mr and pI (Additional file 1). Of the observed proteins, 14.4% were acid (pI below 5) and 26.4% basic proteins (pI above 9). In addition, 27.5% of the proteins had molecular masses of less than 20 kDa.
The hydrophobicity of the proteins was calculated using the GRAVY index, an arbitrary
threshold for high hydrophobicity, and only four proteins had values above 0.2. These
result imply that our extraction technique was somewhat biased against highly hydrophobic
proteins. In Additional file 2, the proteins and all of the respective identified tryptic peptides are listed. Due
to quantitative variability of spectral counting methods in the low abundance range
[17], only proteins with an APEX value of 3,500 or higher in at least one time point were
analyzed further (Additional file 3). This approach resulted in the identification of 375 proteins including 189, 215,
215 and 230 proteins detected at the respective four time points (0 h, 4 h, 6 h, and
8 h) (Table 1 and Figure 2). While low abundance proteins are of interest, they are notoriously difficult to
quantify reliably with shotgun proteomics approaches on most instrument platforms.
The newest LC-MS platforms, e.g. the LTQ Orbitrap Velos, promise to eliminate these
bottlenecks for proteome-wide quantification [18,19].

Figure 2.Venn diagram of proteins detected at 0 h, 4 h and 6 h of fungal growth. Each circle represents the number of proteins detected with APEX expression values
above 3,500 at different time points. The numbers of analyzed and detected proteins
for each time point are shown in Table 1.

About 83% of the 375 proteins analyzed had at least one assigned Gene Ontology (GO)
term from the Biological Process, Molecular Function, and Cellular Component ontologies
(Figure 3 and Additional file 4 and Additional file 5). Most proteins were intracellular including 75 mitochondrial, 92 cytoplasmic, 70
ribosomal, and 45 nuclear proteins. Only thirteen of the 375 proteins were previously
associated with cell wall, plasma membrane or extracellular regions based on their
glycosylphosphatidyl-inositol (GPI) anchor motif or experimental evidence. The large
fraction of intracellular proteins was unexpected, since we applied a mild alkali
extraction method [20] that was previously shown to release alkali-sensitive proteins covalently linked
to glucans in Candida albicans cell walls and also to recover proteins released from lysed cells but retained in
the cell pellets in insoluble or cell surface-bound forms. It remains to be shown
that A. fumigatus immobilizes proteins in its cell wall via glycan linkages. Despite the apparent lack
of enrichment for cell wall proteins, we detected 18 out of 62 proteins previously
associated with the secreted A. fumigatus proteome [21] (Table 2). Notably, fewer than 6% (22 proteins) detected in this study had a predicted signal
peptide or signal anchor sequence. Also, only 20 (3%) putative integral membrane proteins
were identified; all present at very low levels. This was less than expected based
on the total number of putative proteins in the A. fumigatus proteome [2,3], suggesting that extraction of fungal membrane proteins for proteomics analysis remains
a difficult task.

Additional file 4.Cellular localizations of proteins enriched during early development.

Most proteins were involved in translation, respiratory metabolism, amino acid and
carbohydrate biosynthesis, tricarboxylic acid cycle, and other housekeeping functions.
Five common allergens, Asp F3, F8, F9, F12 and F22, and three adhesin-like proteins
were detected. We also found four known virulence factors including cell wall organization
protein Ecm33 (AFUA_4G06820), Mn superoxide dismutase SodB (AFUA_4G11580), homocitrate
synthase HcsA (AFUA_4G10460), and citrate synthase Cit1/McsA (AFUA_6G03590). One protein,
conidial pigment biosynthesis scytalone dehydratase Arp1 (AFUA_2G17580), was implicated
in interactions with the host.

Comparisons with previous studies of the early A. fumigatus proteome showed that different shotgun approaches complement each other with respect
to protein identification (Table 2). We detected 14 out of 26 conidial surface associated proteins [5] and 28 out of 40 most abundant intracellular conidial proteins [6] previously found by 2-D PAGE. Additionally, we found 55 out of 66 immuno-reactive
cytosolic proteins extracted from germinating conidia [8]. Our study also identified 168 out of 231 proteins previously detected in A. fumigatus during early development by iTRAQ [11]. Further comparison with the Cagas et al. study showed that quantification of expression
values using shotgun proteomics methods continues to be a challenge (see below). Some
of these discrepancies can be explained by different time points or score cutoffs
used to define differentially expressed genes and proteins, while others may result
from differences in the proteomics approaches or growth media used.

Proteins expressed at all four stages during early fungal development

Out of 375 proteins, 143 were expressed at all four time points, while the remaining
232 were not detected at one or more time points (Additional file 6). All but ten proteins had an assigned GO biological function (Figure 3). One third of the proteins were ribosomal components or related proteins that function
in translation. The rest had an assigned role in oxidative phosphorylation, amino
acid biosynthesis, gluconeogenesis, and tricarboxylic acid cycle. All 143 proteins
have orthologs in other Aspergillus species, and the majority of them are highly evolutionarily conserved across a broad
range of fungal species. All but twenty proteins were encoded in central regions of
chromosomes (i.e. least 600 Kb from telomeres), which typically are reserved for the
most evolutionary conserved functions such as genome replication, expression, and
central metabolism. Most of the 143 proteins were detected in A. fumigatus in earlier proteomics studies (Table 2). Thus, 70% of them were previously identified using iTRAQ proteomics [11].

Additional file 6.Proteins expressed at all four stages during early development.

Most proteins that were expressed at all four time points showed a moderate increase
in abundance at 4 h, 6 h and 8 h with respect to the 0 h time point. The most abundant
proteins detected at all four time points included conidial hydrophobin Hyp1/RodA
(AFUA_5G09580), allergens Asp F3 (AFUA_6G02280), Asp F8 (AFUA_2G10100) and Asp F22
(AFU A_6G06770), and subunits of the translation elongation factor. Out of 143 proteins,
38 were constitutively expressed at all four time points. These were defined as proteins
with log2 ratios less than 1.5 (see Methods). Four of these constitutively expressed
proteins were characterized as upexpressed in conidia using iTRAQ or 2-D PAGE approaches
[6,11]. Thus, allergen Asp F22 (AFUA_6G06770), Hyp1/RodA (AFUA_5G09580), malate dehydrogenase
(AFUA_6G05210), and zinc-containing alcohol dehydrogenase (AFUA_4G08240) were previously
characterized as conidia-enriched proteins in both of these studies. In contrast,
our analysis showed only a very moderate decrease in their abundance levels at 4 h
or at later stages (Additional file 6). We limited the differential expression analysis comparing developmental time points
to proteins that had at least 4 significant peptides from Peptide/ProteinProphet analysis
at a 5% false discovery rate set.

Dormant conidia enriched proteins (0 h)

To identify proteins enriched at 0 h in comparison to 4 h in A. fumigatus, we analyzed all proteins expressed at 0 h with an APEX score above 3,500. Using
a cutoff of less or equal than −1.5 for log2 expression ratios (4 h/0 h), 52 dormant
conidia-enriched proteins were found. Most of these proteins were not detected at
4 h, 6 h and 8 h (Figure 4). Half of the conidia enriched proteins have no assigned biological function (Figure
3 and Additional file 7), including 17 ‘hypothetical proteins’. The rest tend to be involved in sporulation,
response to oxidative and hypoxic stress, cell wall biosynthesis, and secondary metabolite
biosynthesis. Only one third of the 0 h enriched proteins have no homologs in other
fungi besides the two closest relatives of A. fumigatus: Aspergillus clavatus and Neosartoria fischeri (Aspergillus fischerianus). This is consistent with previous findings that most conidia enriched transcripts
have no assigned biological roles and are lineage specific in other fungi (see [22] for review). Interestingly, small proteins were significantly over-represented in
dormant conidia. Thus, the average M.W. of these proteins was 26,294, which was almost
half the average M.W. of the proteins enriched at the 8 h time point (44,256).

Figure 4.Proteins of high abundance inA. fumigatusconidia. Abundances derived from APEX values ranging from o to 440,000 are displayed in a
heat map generated with the MeV analysis software. More protein information is provided
in Additional file 7 where proteins are listed in the same order.

Out of 52 proteins, 28 have never been previously identified as abundant or over-represented
in A. fumigatus dormant conidia [5,6,8,11]. Using the WoLF PSORT software tool, only two functionally not characterized proteins
(AFUA_1G13670 and GPI-anchored protein AFUA_4G09600) were predicted to localize extracellularly
(Additional file 7). The smallest and most abundant protein detected at 0 h was a protein of unknown
function called Grg1 (AFUA_5G14210). Although Grg1 has not been identified in previous
proteomics studies, its transcripts have been detected in A. fumigatus conidia [23] and shown to be up-regulated in conidia exposed to neutrophils [24]. In A. nidulans, Grg1 transcripts are up-regulated in mycelia exposed to light [25]. Its orthologs in other fungi have been proposed to function as a developmentally
regulated, general stress protein involved in lifespan control [26,27].

Another interesting protein of unknown function enriched at 0 h was ConJ (AFUA_6G03210).
Although ConJ’s biological role is unknown, its transcripts were shown to be upregulated
in the early A. fumigatus transcriptome [11] and during initiation of murine infection [15]. Its ortholog, CON-10, was associated with conidial development in N. crassa. Transcripts of CON-10 were shown to accumulate in vegetative mycelia upon blue light
exposure and during conidial development [28]. Both Grg1 and ConJ were computationally predicted to have nuclear localization.
Another 0 h-enriched protein of note was the pigment biosynthesis scytalone dehydratase
Arp1 (AFUA_2G17580). Arp1 is encoded by the six-gene pigment biosynthesis cluster,
which also encodes proteins that have been earlier associated with conidia. The conidial
pigment, melanin, has been shown to contribute to fungal virulence in a murine model
[29] and to modulate the host cytokine response by masking specific ligands on the A. fumigatus cell surface [30,31]. 1,8-dihydroxynaphthalene-melanin was shown to inhibit phagolysosomal acidification
[32].

A few heat shock proteins and other chaperons involved in maturation of protein complexes
were also upexpressed in the A. fumigatus dormant conidia. Many of these proteins have never been previously associated with
A. fumigatus spores including heat shock protein Scf1/Awh11 (AFUA_1G17370), nascent polypeptide-associated
complex subunit Egd2 (AFUA_6G03820), calnexin ClxA (AFUA_4G12850), and Hsp70 chaperone
BipA (AFUA_2G04620). The exact biological role of Scf1 is unknown. It is a possible
target of transcription factor CrzA, which is a downstream effector of the calcineurin
signaling pathway and regulates conidial germination, hyphal growth, and pathogenesis
in A. fumigatus[33,34]. In A. nidulans, Scf1 transcription is repressed by StuA, which also regulates multicellular complexity during asexual reproduction, ascosporogenesis
and multicellular development during sexual reproduction [35]. Scf1 also has a S. cerevisiae ortholog, HSP12, which is a plasma membrane protein involved in maintenance of membrane
organization under stress and in response to heat shock, oxidative stress, and osmotic
stress [36].

Similarly, chaperons ClxA and BipA are involved in unfolded protein response and possibly
ER stress in fungi. In filamentous fungi, calnexin is involved in N-glycan-dependent
quality control of folding of cell-wall-targeted glycoproteins [37,38]. Glycosylation is a conserved posttranslational modification that is essential for
cell wall function [39]. A recent 2-D PAGE study showed that overexpression of calnexin and a putative HSP70
chaperone is activated by the deletion of the cwh41 gene encoding glucosidase I in A. fumigatus, which also leads to ER stress and possibly activates the ER-associated degradation
[40].

Among other unusual findings was the detection of a putative transcription factor,
HapB (AFUA_2G14720) and histones H3 and H4.1 (AFUA_1G13780 and AFUA_1G13790). Orthologs
of HapB have been shown to function in regulation of carbohydrate metabolism in A. nidulans[41] and sporulation in yeast [42], and the subunit HapE of the CCAAT-binding complex was previously identified in dormant
conidia [6]). This complex was shown to be a key regulator of redox homeostasis in A. nidulans[43]. Histones H3 and H4.1 have been implicated in sporulation in S. cerevisiae[44].

In addition to 28 novel conidia enriched proteins, 24 proteins including known virulence
factors were discovered in previous proteomics studies (Table 2) [5,6,8,11]. Nine of the 0 h-enriched A. fumigatus proteins were identified as overexpressed in dormant conidia vs. mycelium by Teutschbein
and colleagues using 2-D PAGE [6]. Among these were Mn superoxide dismutase SodB (AFUA_4G11580) and endopeptidase Pep2
(AFUA_3G11400), two conidial pigment biosynthesis proteins, Ayg1 and Arp2 (AFUA_2G17550
and AFUA_2G17560), a putative methyltransferase (AFUA_8G00550), and 2-methylcitrate
synthase McsA (AFUA_6G03590). SodB, also known as allergen Asp F6, was also detected
in the secreted A. fumigatus proteome [21]. SodB is considered a putative virulence factor, because it detoxifies superoxide
anions and its transcripts are up-regulated in conidia exposed to neutrophils and
by the oxidative agent menadione [24]. However, a triple deletion mutant (sod1sod2sod3) did not show attenuation in virulence[45]. Endopeptidase Pep2 is a conidia surface-associated protein [5], whose transcripts are up-regulated in conidia exposed to neutrophils [5,6,24,46]. Alb1, not identified in this study, and McsA have been characterized putative virulence
factors in A. fumigatus. The alb1 gene is also involved in conidial morphology and resistance to oxidative stress [47]. AFUA_8G00550 is encoded by a pseurotin A biosynthesis cluster [48]. It is induced during hypoxia and over-represented in conidial [6,49].

Additionally, three of 0 h-enriched proteins were previously identified as highly
abundant in the conidial proteome by the same authors [6]. The list includes a hypothetical protein (AFUA_6G12000), an Asp hemolysin-like protein
(AFUA_4G02805), and mannitol-1-phosphate dehydrogenase MpdA (AFUA_2G10660). AFUA_6G12000
is the second most abundant protein at 0 h and is unique to A. fumigatus and its close relative, N. fischeri. The functional role of Asp hemolysin-like protein (AFUA_4G02805) is not yet known.
Its paralog, Asp hemolysin (AFUA_3G00590), was recently identified as a major secreted
protein expressed in resting and germinating conidia and during hyphal development
[21]. Both proteins belong to the protein family of aegerolysins, which includes a large
number of bacterial and fungal proteins that function in sporulation and development.
MpdA protein is induced by heat shock and reacts with rabbit immunosera exposed to
A. fumigatus germling hyphae [50]. In A. niger, the mpdA gene expression is increased in the sporulating mycelium [51,52]. This indicates that mannitol biosynthesis may be developmentally regulated in aspergilli.
Mannitol itself has been shown to play a key role in ensuring the stress tolerance
of A. niger conidiospores [52].

Expanding conidia-enriched proteins (4 h)

Out of 215 proteins detected at the 4 h time point, 85 were identified as up-expressed
at 4 h in comparison to 0 h in A. fumigatus conidia. Remarkably, 25 of these proteins (29%) were not detected in dormant conidia,
while 44 (52%) were also up-expressed at 6 h and 8 h in comparison to 0 h. This is
consistent with the view that the dramatic shift in protein expression associated
with conidial expansion happens between 0 h and 4 h time points. Most proteins (85%)
had an assigned GO biological function, with translation and tricarboxylic acid cycle
being the most common ones (Additional file 8). Almost half of 4 h enriched proteins (52 out of 85 proteins) were previously identified
in the A. fumigatus conidial proteome [5,6,8,11] (Table 2).

While the majority of 4 h enriched proteins were intracellular, the list also includes
five cell wall proteins such as cell wall organization protein Ecm33 (AFUA_4G06820),
which was earlier implicated in conidial germination, antifungal drug resistance,
and hypervirulence [53,54]. Four GPI-anchored proteins were also identified including beta-1,3-endoglucanase
EglC (AFUA_3G00270), which has been implicated in cell wall organization and biosynthesis
[55]. EglC was also detected in the A. fumigatus immunosecretome, secreted proteome and in germinating conidia [8,21,56].

Among the most abundant proteins in expanding conidia were allergens Asp F8/60 S ribosomal
protein P2 (AFUA_2G10100) and Asp F3 (AFUA_6G02280), several cytosolic ribosomal subunits,
and the putative cell cycle regulator Wos2 (AFUA_5G13920). A. fumigatus Wos2 has been shown to be recognized by immunosera from rabbits exposed to conidia
[50], while its orthologs function in regulation of the cell cycle in A. niger and of telomerase activity in yeast [57,58]. The predominance of known allergens and other immunoreactive proteins in expanding
conidia is consistent with previous studies. This initial stage of spore germination,
also known as “swelling,” triggers the recruitment of host inflammatory cells.

Another immunoreactive protein enriched at 4 h was CipC (AFUA_5G09330), which was
shown to react with immunosera from rabbits exposed to A. fumigatus conidia [50]. It has never before been associated with the conidial proteome, but described a
major hyphal-specific protein [59]. Proteomic evidence indicated that CipC is a secreted protein [5]. Its exact function is unknown, although it was suggested that it is involved in
competitive interactions between bacteria and aspergilli. CipC was associated with
the hyphal morphotype that enables invasive growth during infection. Proteome analysis
of A. nidulans identified its close homolog CipC (but not AFUA_5G09330) as a protein associated
with the response to stress and the antibiotic concanamycin A [60].

Amino acid biosynthesis proteins were also abundant at 4 h including homocitrate synthase
HcsA (AFUA_4G10460), which has been implicated in A. fumigatus virulence and is considered a possible antifungal drug target [61]. HcsA is also expressed at 6 h and 8 h. The protein is required for lysine biosynthesis
and has been shown to be induced by heat shock [62]. This virulence factor has not been associated previously with A. fumigatus conidial proteome, however, its transcript is known to be highly induced during conidial
germination [12].

Among unusual findings was the discovery of regulatory protein suAprgA1 (AFUA_3G09030),
which has not been previously associated with conidia. Although its exact function
is unknown, it is a highly conserved protein with putative homologs in mammals, fungi
and protozoa. Its orthologs have been shown to function in aerobic respiration in
S. cerevisiae and in regulation of penicillin biosynthesis in Aspergillus nidulans[63]. In contrast, its homolog regulates the RNA-binding activity of a protein that guides
RNAs during the mitochondrial RNA editing process in Trypanosoma brucei[64].

Additionally, some proteins were detected at 0 h and 4 h time points such as cell
wall integrity signaling protein Pil1 (AFUA_6G07520). It is the only one signaling
protein detected in the early A. fumigatus proteome. Its ortholog has been detected in the A. nidulans proteome at 0 h and 1 h time points [65]. It localized to the conidial periphery and in punctate structures in mycelia [65,66]. A. fumigatus Pil1 is similar to yeast sphingolipid long chain base-responsive protein PIL1, which
is a primary component of large immobile cell cortex structures associated with endocytosis.
PIL1 null mutants show activation of Pkc1p/Ypk1p stress resistance pathways in S. cerevisiae[67].

Early germ tube-enriched proteins (6 h)

Out of 215 proteins found in hyphae with early germ tubes, 127 (59%) were identified
as over-expressed in comparison to dormant conidia in A. fumigatus. The vast majority (94%) of 6 h enriched proteins had an assigned GO biological function
(Additional file 9). Most common functions included translation, ATP synthesis coupled electron transport,
amino acid biosynthesis, gluconeogenesis, and tricarboxylic acid cycle. Almost half
were ribosomal components and proteins that function in translation. The proteins
appear to be evolutionarily conserved across a broad range of fungal species as well
as in other eukaryotes including humans. All but four proteins of the 127 proteins
(97%) were encoded by genes located in central regions of chromosomes (>300 Kb from
telomeres), which on average harbor only 85% of A. fumigatus genes. Only two proteins were annotated as “hypotheticals”, because they shared no
sequence similarity with any characterized protein or domain in public databases.
Both proteins were only detected at 6 h.

Eighty five of the 127 proteins (67%) were also enriched in early germ tubes in comparison
to expanding conidia, reflecting continuous exponential increase in the biosynthetic
capacity during these three developmental stages. The most abundant proteins among
those were translation elongation factor subunits, components of the cytosolic ribosome,
thiazole biosynthesis enzyme ThiF (AFUA_6G08360), glyceraldehyde 3-phosphate dehydrogenase
GpdA (AFUA_5G01970), and plasma membrane H + −ATPase Pma1 (AFUA_3G07640). ThiF has
not been detected in the A. fumigatus proteome prior to this study. The ThiF yeast ortholog, THI4, has been shown to catalyze
formation of a thiazole intermediate during thiamine biosynthesis and to be required
for mitochondrial genome stability in response to DNA damaging agents [68]. GpdA has been shown to react with immunosera from rabbits exposed to A. fumigatus conidia [50].

Some of the 6 h enriched proteins may have important roles in establishing mammalian
infection. Thus homocitrate synthase HcsA (AFUA_4G10460) and superoxide dismutase
SodA (AFUA_5G09240), previously implicated in the initiation of infection, are up-expressed
at this stage. HcsA has not been associated with A. fumigatus conidia or germling hyphae in proteomics studies. Furthermore, transcripts for six
of these proteins were up-regulated in A. fumigatus germlings during initiation of murine infection [15]. The list includes ThiF, mentioned above, cell wall glucanase BtgE (AFUA_8G05610),
superoxide dismutase SodA (AFUA_5G09240), pyridoxine biosynthesis protein PyroA (AFUA_5G08090),
and pyruvate carboxylase (AFUA_4G07710). BtgE is a covalently bound cell wall protein
with a predicted role in degradation of glucans.

In contrast to 0 h enriched proteins, there is a much higher degree of correlation
between 6 h enriched proteins and the proteins identified during early developmental
stages in previous A. fumigatus proteomics studies (Table 2). Thus, 78 out 127 (61%) the latter were previously detected at 0 h, 4 h, 8 h and
16 h [30], including 15 and 13 proteins up-expressed at 8 h and 16 h of mycelial growth. Moreover,
transcripts of 105 and 91 proteins (83% and 72%, respectively) were shown to be up-regulated
at 8 h and 16 h respectively. Also, 14 and five of our germling hyphae-enriched proteins
were previously identified as highly abundant in conidia and overrepresented in conidia
in comparison to mycelia, respectively [6].

Pre-septation hyphae-enriched proteins (8 h)

A total of 119 proteins were up-expressed at 8 h of fungal growth in comparison to
dormant conidia (Additional file 10). Of those, 103 (87%) had an assigned GO biological function (Figure 3). At least, 26 proteins were involved in translation either as ribosomal subunits
or as components of a translation elongation factor. Similar to 6 h enriched proteins,
most 8 h enriched proteins were evolutionary conserved, and all but four were encoded
by central chromosomal regions. All but two of the 119 proteins had orthologs in other
aspergilli [3].

The list of the most abundant 8 h enriched proteins included allergen Asp F8/60 S
acidic ribosomal protein P2 (AFUA_2G10100), a protein of unknown function (AFUA_1G06580),
and a mitochondrial cytochrome c subunit (AFUA_2G03010). More than half of the enriched
proteins were also overexpressed at 6 h and six proteins showed a pattern of exponential
increase from 0 h through 8 h of fungal growth. The latter included allergen Asp F8/(AFUA_2G10100),
two cell wall proteins (AFUA_4G08960 and AFUA_8G05610), nucleolar pre-rRNA processing
protein Nop58 (AFUA_3G13400) and a subunit of a eukaryotic translation initiation
factor (AFUA_4G03860). One GPI-anchored protein (AFUA_8G05610) is a putative adhesin,
while the other (AFUA_3G00270) is cell wall glucanase BtgE. Notably, BtgE transcripts
have been shown to be up-regulated during initiation of murine infection by A. fumigatus[15]. Additionally, six pre-septation hyphae-enriched proteins were detected previously
in the secreted A. fumigatus proteome including Cu,Zn superoxide dismutase SodA (AFUA_5G09240) and extracellular
cell wall glucanase Crf1/allergen Asp F9 (AFUA_1G16190). Similar to BtgE, SodA was
previously detected in conidia and its transcripts were up-expressed in germlings
during initiation of murine infection in A. fumigatus[15].

Conclusions

The observed temporal expression patterns suggest that germination of A. fumigatus conidia involves dramatic changes in protein abundance levels. Some of the 375 identified
proteins may represent novel antigens and stage-specific biomarkers of colonization,
infection or treatment efficacy. Developmental stage candidate biomarkers include
the following proteins: (0 h) Grg1, AFUA_6G12000; (4 h) Hsp90 binding co-chaperone
Wos2 and a CipC family protein; (6 h) 40 S ribosomal protein S19 and the conserved
protein AFUA_2G10580; and (8 h) telomere and ribosome associated protein Stm1 and
glycine-rich RNA-binding protein. Additionally, we found that the A. fumigatus conidial proteome is dominated by small, lineage-specific proteins that may play
key roles in host-pathogen interactions and in transmitting environmental signals
that control conidial germination. Small proteins are more difficult to study than
larger proteins using traditional biochemical and molecular methods. Our results show
that shotgun proteomics can facilitate functional characterization of these interesting
targets, which can be exploited to make the fungus more vulnerable to the host immune
system.

Methods

A. Fumigatus growth and harvest

3 x 108 per 100 ml of A. fumigatus CEA10 conidia were washed with H2O and inoculated into Glucose Minimal Media and incubated at 37°C at 200 rpm for 4,
6 and 8 h. For the 0 h time point, freshly harvested conidia were used. Cell wall
protein extraction was conducted using a modified version of a previously described
protocol [20,69]. The cells were harvested using Corning 500 ml bottle top filter and rinsed with
cold sterile water and then with 10 mM Tris–HCl, pH7.5.

Protein digestion

The frozen conidia pellet was ground to a fine powder using a mortar and pestle. Cells
were re-suspended in 10 mM Tris–HCl, pH 7.5 (25 ul/mg) in the presence of a protease
inhibitor cocktail (Roche, complete Mini EDTA-free Protease inhibitor cocktail). Soluble
proteins, likely to be primarily of intracellular origin, were removed by washing
the insoluble fraction three times with 1 M NaCl, centrifuging at 300 rpm for 10 min
at 4°C between each wash. The insoluble fraction was then twice extracted for 5 min
at 100°C with SDS extraction buffer (50 mM Tris–HCl, pH 7.8, 2%SDS, 100 mM NaEDTA,
and 40 mM β-mercaptoethanol). The SDS treated insoluble fraction was washed three
times with water and spun at 300 rpm for 5 min between each wash, followed by incubation
with 30 mM NaOH at 4°C for 17 h with gentle shaking. The reaction was stopped by addition
of neutralizing amounts of acetic acid. Overnight dialysis of the released proteins
at 4°C was carried out. The proteins were precipitated by adding 9 volumes of 100%
methanol buffer (100% methanol, 50 mM Tris HCl, pH 7.8), incubating at 0°C for 2 h,
and centrifugation at 13,000 rpm for 10 min at 4°C. The pellet was washed twice with
90% methanol buffer (90% methanol, 50 mM Tris HCl pH 7.8) and air dried. The pellet
was dissolved in 10 mM Tris HCl pH 7.5. The protein concentration was determined according
to the method of Bradford using BIO-RAD protein assay (BIO-RAD Lab., U.K.) [70]. The ten analyzed samples contained between 35 and 70 μg protein, suggesting that
this extraction procedure did not result in retention of large amounts of intracellular
protein. They were processed using filter-aided sample preparation (FASP) and suitable
for downstream mass spectrometric analysis [71]. In this way, in-solution digestion was carried out in the filter device, where denatured
proteins were digested under the condition of maintaining the activity of the trypsin
without a carboxyamidomethylation step to modify cysteine residues. The entire protein
digests checked in SDS-PAGE gels for completion of digestion were analyzed by LC-MS/MS
to identify A. fumigatus proteins.

LC-MS/MS

LC-MS/MS analysis was performed with a LTQ ion trap mass spectrometer (Thermo-Finnigan,
San Jose, CA) equipped with a Finnigan nESI source. An Agilent 1100 series solvent
delivery system (Agilent, Palo Alto, CA) was interfaced with the LTQ instrument to
deliver samples to a peptide trapping cartridge (CapTrap, Michrom BioResources, Auburn,
CA), followed by a reversed-phase column. Peptides were eluted from the C18 cartridge and separated on the BioBasic C18 column (BioBasic C18, 75 μm × 10 cm, New Objective, Woburn, MA) for 85 min run [53 min binary gradient
run from 97% solvent A (0.1% formic acid) to 80% solvent B (0.1% formic acid, 90%
AcCN) at a flow rate of 350 nl/min.] Mass spectra were acquired in automated MS/MS
mode, with the top five parent ions selected for fragmentation in scans of the m/z
range 300–1,500 and with a dynamic exclusion setting of 90 s, deselecting repeatedly
observed ions for MS/MS as previously provided [72].

MS data analysis

MS and MS/MS sequences obtained from LC-MS/MS experiments were searched against the
latest release of the NCBI A. fumigatus proteome (WGS AAHF01000001-AAHF01000019) using the search engine Mascot v. 2.3.2
(Matrix Science, London, UK). LTQ peak lists were created with Mascot Daemon using
the data import filter lcq_dta.exe from XCaliber v.2.2 (Thermo electron), which coverts
binary.raw files into peak list.dta files. The data were retrieved with search parameters
set as follows: enzyme, trypsin; allowance of up to one missed cleavage peptide; MS
tolerance ±1.4 Da and MS/MS tolerance ± 0.5 Da; no modification of cysteine and methionine
oxidation when appropriate with auto hits allowed only significant hits to be reported.
The protein identifications were accepted as significant when a Mascot protein score
>75 and at least one peptide e-value <0.01 were reported. To accept a Mascot score
between 40 and 75, a protein had to be identified as least two times with at least
two peptide e-value <0.05 each. Using a randomized decoy database and a default significance
threshold of 0.05 in Mascot, the false-positive rate for peptides identified by LC-MS/MS
was 1.6%. Following file conversion into the ‘mzXML’ format, MS data were re-scored
using the algorithms PeptideProphet™ and ProteinProphet™ [73]. The data is available in the PRIDE database [74] (http://www.ebi.ac.uk/pridewebcite) under accession numbers [19312–19315].

Calculation of protein abundance estimates using the APEX method

The LC-MS/MS data from biological replicates (duplicates for 4 h and 6 h time points;
triplicates for 0 h and 8 h time points) were combined to calculate absolute protein
expression (APEX) values using a computationally modified spectral counting approach
developed by Lu et al. [10] and converted into a software application by Braisted et al. termed the APEX quantitative
proteomics tool v1.1 [16]. Briefly, the XML spectral data files were converted into Peptide/Protein Prophet
probabilities, and Oi correction factors based on probability of peptide detection determined to adjust
the protein quantities based on spectral counts. Default settings for peptide physicochemical
properties were used to determine Oi values. A normalization factor of 2.0 × 106 was used to convert the APEX scores into estimates of protein molecules per cell.
The protein FDR was set at 1% to eliminate proteins identified at a confidence level
lower than 99%. To apply a higher stringency level to the evaluation of differential
protein abundances comparing the four time points, only proteins with the following
filter criteria were included in the abundance analysis: (1) a total significant peptide
count of at least 4 according to Mascot and APEX data and a significant unique peptide
count in Mascot of at least 2 or (2) an APEX scores higher than 3,500. To identify
differentially expressed proteins, Log2 ratios were used to measure relative changes
in expression level at 4 h, 6 h and 8 h time points with respect 0 h. To add another
level of quantification stringency, proteins were considered differentially expressed
if their APEX expression values were above 3,500 and their corresponding log2 ratios
were greater than 1.5 or less than −1.5.

For the prediction of N-terminal signal peptides and transmembrane regions, acquired
amino acid sequences of all proteins were searched with the algorithms SignalP and
TMHMM (http://www.cbs.dtu.dkwebcite). For subcellular locations, a WoLF PSORT software (freely available at wolfpsort.org)
was used to predict the subcellular localization. Gene Ontology (GO) terms were downloaded
from AspGD (http://www.aspergillusgenome.orgwebcite) [75]. The GO Slimmer tool (http://amigo.geneontology.orgwebcite) was used to obtain higher level broader parent terms GO molecular function and cellular
localization predictions also known as GO Slim terms.

Authors' contributions

MM and RP initiated and coordinated this study and contributed to the preparation
of the manuscript. RP selected the proteomics approach, MS conducted the proteomics
analysis. MS and NDF performed the analysis and interpretation of data and drafted
the manuscript, and SH cultured A. fumigatus and prepared all protein extracts. RDF, SNP, WCN, SC and DP have been involved in
revising of the manuscript and made contributions to the study conception and design.
All authors have read and approved the final manuscript.

Competing interests

The author(s) declare that they have no competing interests.

Acknowledgements

We would like to thank Shih-Ting Huang and Nikhat Zafar for their superb bioinformatics
assistance. This project has been funded in whole or part with federal funds from
the National Institute of Allergy and Infectious Diseases, National Institutes of
Health, Department of Health and Human Services under contract numbers N01-AI-15447,
N01-AI30071 and HHSN272200900007C.

Lottspeich F: Top Down and Bottom Up Analysis of Proteins (Focusing on Quantitative Aspects). In Protein and Peptide Analysis by LC-MS: Experimental Strategies. Edited by Letzel T. Cambridge, United Kingdom: The Royal Society of Chemistry; 2011:1-10.